Drug Discovery Assay for Modulators of HIF-Prolyl Hydroxylase Activity

Abstract

The present invention relates to a high throughput assay for determining HIF 1α prolyl hydroxylation.

1 . A method for determining hypoxia-inducible factor prolyl hydroxylation activity comprising:
 a. incubating a mixture comprising a biotinylated proline-containing substrate and an HPH enzyme in a suitable buffer for a sufficient time to allow hydroxylation of said substrate;   b. quenching said reaction with a composition comprising a VBC complex attached to a GST (GST-VBC), streptavidin-labeled allophycocyanin and a fluorescently labeled anti-GST antibody for a time sufficient to allow the VBC complex to interact with said proline-containing substrate; and   c. determining the amount of interaction of said GST-VBC with said substrate.   
     
     
         2 . The method of  claim 19  wherein said HPH enzyme is selected from the group consisting of a HPH 1, HPH 2, and an HPH 3 enzyme. 
     
     
         3 . The method of claim of  claim 2 , wherein said HPH enzyme is a mammalian HPH enzyme. 
     
     
         4 . The method of claim of  claim 3 , wherein said HPH enzyme is a human HPH enzyme. 
     
     
         5 . The method of claim of  claim 3 , wherein said HPH enzyme is a monkey HPH enzyme. 
     
     
         6 . The method of claim of  claim 3 , wherein said HPH enzyme is a rhesus monkey HPH enzyme. 
     
     
         7 . The method of  claim 3 , wherein said HPH enzyme has a sequence selected from the group consisting of SEQ ID NO:1; SEQ ID NO:2, SEQ ID NOS:3; SEQ ID NO:4, SEQ ID NO: 5, and SEQ ID NO:6. 
     
     
         8 . The method of  claim 3 , wherein said HPH enzyme is rhesus monkey HPH having a sequence of SEQ ID NO:3. 
     
     
         9 . The method of  claim 1 , wherein said proline-containing substrate is a peptide comprising the sequence LXXLAP, wherein P in said substrate is hydroxylated by the HPH and X represents any amino acid. 
     
     
         10 . The method of  claim 1 , wherein said proline-containing substrate is a peptide comprising the sequence LAPY, wherein P in said substrate is hydroxylated by the HPH. 
     
     
         11 . The method of  claim 19  wherein said proline-containing substrate is a peptide comprising the sequence LAPYI, wherein P in said substrate is hydroxylated by the HPH. 
     
     
         12 . The method of  claim 1 , wherein said proline-containing substrate is a peptide comprising the sequence of LAPYIP or LAPYIG, wherein P in said substrate is hydroxylated by the HPH. 
     
     
         13 . The method of  claim 1 , wherein said proline-containing substrate is a peptide comprising a sequence selected from the group consisting of DLDLEALAPYIPADDDFQL, DLDLEMLAPYIPMDDDFQL, DLDLEMLAPYIGMDDDFQL, and or DLDLEALAPYIGADDDFQL (SEQ ID NO______). 
     
     
         14 . The method of  claim 1 , wherein the reaction mixture in step a further comprises 2α-ketoglutarate as a substrate. 
     
     
         15 . The method of  claim 1  wherein said fluorescently labeled anti-GST antibody comprises a fluorescent label selected from the group consisting of europium (Eu), dysprosium (Dy), lanthanum (La), gadolinium (Gd), cerium (Ce), samarium (Sm), yttrium (Y), neodymium (Nd), terbium (Tb), praseodymium (Pr), erbium (Er), thulium (Tm), ytterbium (Yb), scandium (Sc), promethium (Pm), holmium (Ho), and lutetium (Lu). 
     
     
         16 . The method of  claim 1 , wherein the detecting comprises a time resolved homogeneous fluorescence assay. 
     
     
         17 . The method of  claim 16 , wherein said fluorescently labeled anti-GST antibody is labeled with Europium. 
     
     
         18 . The method of  claim 17 , wherein said detecting comprises detecting the signal at 665 nm and the signal at 620 nm, wherein the ratio of the signal at 665:620 nm proportional to the amount of peptide interaction with said GST-VBC. 
     
     
         19 . The method of  claim 1 , wherein said assay is performed in the presence of a modulator of HPH activity. 
     
     
         20 . The method of  claim 1 , wherein said method is a high throughput screening assay in a multi-well plate format. 
     
     
         21 . A method of screening for a modulator of HIF prolyl hydroxylation., comprising the steps of:
 a. incubating a mixture comprising an isolated HIF-specific prolyl hydroxylase (HPH) protein and a peptide substrate of HPH hydroxylase enzymes, under conditions whereby a proline residue on said substrate is hydroxylated, and   b. quenching said reaction with a composition comprising a VBC complex attached to a GST (GST-VBC), streptavidin-labeled allophycocyanin and a fluorescently labeled anti-GST antibody for a time sufficient to allow the VBC complex to interact with said proline-containing substrate; and   c. detecting a resultant prolyl hydroxylation of the substrate using time resolved homogeneous fluorescence;   wherein the incubating step is performed in the presence and absence of candidate modulator of prolyl hydroxylation wherein an increase in the prolyl hydroxylation in the presence of said candidate modulator is indicative of said modulator being an enhancer of prolyl hydroxylation and a decrease in the prolyl hydroxylation in the presence of said candidate modulator is indicative of said modulator being an inhibitor of prolyl hydroxylation.   
     
     
         22 . The method of  claim 21 , wherein said HPH is selected from the group consisting of a protein of SEQ ID NO:1, SEQ ID NO:2 and SEQ ID NO: 3. 
     
     
         23 . A method of determining HIF prolyl hydroxylation activity, comprising the steps of:
 a. incubating a mixture comprising an isolated HIF-specific prolyl hydroxylase (HPH) protein from  M. mulatta  and a peptide substrate having a sequence of biotin-DLDLEMLAPYIPMDDDFQL, under conditions whereby a proline residue on said substrate is hydroxylated, and   b. quenching said reaction with a composition comprising a VBC complex attached to a GST (GST-VBC), streptavidin-labeled allophycocyanin and an europium-labeled anti-GST antibody for a time sufficient to allow the VBC complex to interact with said proline-containing substrate; and   c. detecting a resultant prolyl hydroxylation of the substrate using time resolved homogeneous fluorescence at an excitation wavelength of 330 nm; and emission at wavelengths 620 and 665 nm.   
     
     
         24 . The method of  claim 23 , wherein said incubating step is performed in the presence and absence of candidate modulator of prolyl hydroxylation wherein an increase in the prolyl hydroxylation in the presence of said candidate modulator is indicative of said modulator being an enhancer of prolyl hydroxylation and a decrease in the prolyl hydroxylation in the presence of said candidate modulator is indicative of said modulator being an inhibitor of prolyl hydroxylation. 
     
     
         25 . The method of  claim 23  wherein said reaction mixture comprises a reaction buffer containing 20 mM MOPS (pH 6.5), 1.5 mM magnesium chloride, 5 mM potassium chloride, 0.1% bovine serum albumin, 1 mM TCEP, 10 μM ferrous ammonium sulfate, and 2 mM sodium ascorbate, said peptide substrate, α-ketoglutarate and, HPH enzyme.